In order to detect a substance in a biological sample, a method utilizing a substance that specifically binds to the former substance (substance to be detected) has been widely used. For example, immunoassays utilizing antigen-antibody reactions and nucleic acid hybridization assays utilizing hydrogen bonds between nucleic acid chains are known. For example, in the immunoassay, an antigen, when the substance to be detected is an antibody, or in contrast, an antibody, when the substance to be detected is an antigen, is immobilized on a carrier such as a microplate, microbeads, or a sensor chip, and after the reaction with the substance to be detected, the presence or absence or the degree of the antigen-antibody reaction is measured.
As general means for the immobilization, for example, hydrophobic bonding and covalent bonding are known. In the “hydrophobic bonding”, a carrier and a protein that specifically binds to a substance to be detected (hereinafter, may be referred to as “specific protein”) are bound to each other by interaction between the hydrophobic surface of the carrier and the hydrophobic moiety of the specific protein. This is convenient from the point of not needing specific reagents. However, such binding is usually weak. In the case where the hydrophobic bonding is applied to, for example, enzyme-linked immunosorbent assay (ELISA), the protein is detached from the carrier during, for example, a washing procedure after binding in many cases. Furthermore, in the case where a specific protein is bound to a carrier by hydrophobic bonding, the function of the protein may be lost completely or partially in many cases. The “covalent bonding” utilizes interaction between functional groups (e.g., amino groups) of a specific protein and functional groups (e.g., carboxyl groups) provided on the surface of the carrier, and is strong. However, after a specific protein is bound to a carrier by covalent bonding, the function of the protein is lost completely or partially in many cases, like the hydrophobic bonding.
In addition to the hydrophobic bonding and the covalent bonding, known is a method for fusing a plurality of histidine molecules to terminals of protein molecules and binding the fusion protein having the histidine tags to, for example, a basal plate, such as a protein chip, having a surface provided with nickel. The interaction between the histidine tags and nickel ions is, however, not very strong, and nickel ions are known to non-specifically bind to a variety of biological molecules.
Alternatively, a method of binding a protein to a carrier using the binding ability of avidin or streptavidin to biotin (vitamin H) has been developed. Avidin is a glycoprotein derived from albumen and extremely strongly binds to biotin. The interaction between avidin and biotin is one of the strongest non-covalent bonds (Green, (1975), Adv Protein Chem, 29: 85-133). Meanwhile, streptavidin is an avidin-like protein derived from actinomycetes and also strongly binds to biotin. Biotin is a molecule having a low molecular weight of 244 and can be easily bound to various biological molecules, such as proteins, nucleic acids, lipids, or sugar chains, using, for example, commercially available kits and also hardly affects the properties of biotinylated molecules. The interaction of (strept)avidin-biotin, because of its high binding force, has been widely used, for example, for detection of antigens and antibodies in the fields of molecular biology and biochemistry (Green, (1990), Methods Enzymol, 184: 51-67). In the case of binding a protein to a carrier using avidin or (strept)avidin, (strept)avidin is immobilized to a basal plate such as a microplate through covalent bonding or hydrophobic bonding, and then a biotinylated protein is bound thereto. Furthermore, a technique of immobilizing basal plate-biotin-avidin-biotin-desired protein in this order is also reported, in which avidin is bound to a basal plate provided with biotin by avidin-biotin binding, and then a biotinylated desired protein is bound to another biotin pocket of the avidin (JP No. H4-236353 A (1992)). A subject substance can be detected using a plate on which a specific protein is immobilized by such a technique.
In such a specific binding assay system using a solid phase on which a protein that specifically binds to a substance, such as an antigen or antibody, to be detected is bound by hydrophobic bonding or covalent bonding, non-specific binding, which causes a background signal and thus should be reduced, is generally a severe problem. In order to solve this problem, the following methods have been proposed for example: a method of adding an extract of a bacterium component to a reagent for detection (JP No. S59-99257 A (1984)); a method of adding a culture component of host cells containing a vector of the same species as that used in production of a recombinant protein capable of specifically binding to a substance to be detected and the vector not containing the gene encoding the protein to a sample (JP No. H8-43392 A (1996)); and a method of heat-treating an aqueous extract from cells of the same species as that producing a recombinant protein capable of specifically binding to a substance to be detected, and the cell not containing this protein, and then adding water-soluble fraction of the heated aqueous extract to a sample (JP No. 2004-301646 A). These methods show some effects on inhibition of non-specific binding.
The assay utilizing the avidin-biotin binding has also a big problem with background signals, like the assay using a solid phase on which a protein that specifically binds to a substance to be detected is bound by, for example, hydrophobic bonding or covalent bonding. Countermeasures have been proposed for solving this problem are, for example: a method in which a sample is put into contact with a solid phase to which inactivated (strept)avidin is bound and then contact with a solid phase to which active (strept)avidin is bound (JP No. H8-114590 A (1996)); a method in which a biotinylated substance is bound to an avidin-bound solid phase, and then this is put into contact with a conjugate of polyethylene glycol and biotin (JP No. H11-211727 A (1999)); and a method in which a biotin-containing solution is put into contact with a solid phase (JP No. 2002-48794 A), in addition to the above-mentioned methods for preventing non-specific binding. Unfortunately, all the methods exhibit insufficient practical advantages.